IgA Plasmablasts Research Articles

B cells critical for outcome in high grade serous ovarian carcinoma.

Recent work has shown evidence for the prognostic significance of tumor infiltrating B cells (B-TIL) in high grade serous ovarian carcinoma (HGSOC), the predominant histological subtype of ovarian cancer. However, it remains unknown how the favorable prognosis associated with B-TIL relates to the current standard treatments of primary debulking surgery (PDS) followed by chemotherapy or (neo-)adjuvant chemotherapy (NACT) combined with interval debulking surgery. To address this, we analyzed the prognostic impact of B-TIL in relationship to primary treatment and tumor infiltrating T cell status in a highly homogenous cohort of HGSOC patients. This analysis involved a combined approach utilizing histological data and high-dimensional flow cytometry analysis. Our findings indicate that while HGSOC tumors pre-treated with NACT are infiltrated with tumor-reactive CD8+ and CD4+ TIL subsets, only B-TIL and IgA plasma blasts confer prognostic benefit in terms of overall survival. Importantly, the prognostic value of B-TIL and IgA plasma blasts was not restricted to patients treated with NACT, but was also evident in patients treated with PDS. Together, our data point to a critical prognostic role for B-TIL in HGSOC patients independent of T cell status, suggesting that alternative treatment approaches focused on the activation of B cells should be explored for HGSOC.

The immune system of children: the key to understanding SARS-CoV-2 susceptibility?

The immune system of children: the key to understanding SARS-CoV-2 susceptibility?

Human B cell responses to a live attenuated cholera vaccine

Abstract Vibrio cholerae is a bacterial pathogen which causes cholera, a severe acute diarrheal disease. A symptomatic incident of cholera can lead to long term protection against subsequent exposure; however, the generation of immunity and mechanisms of protection are undetermined. A thorough understanding of the immune response to V. cholerae is needed to identify parameters which are key for the generation and maintenance of immunity. To approach this, we utilized a live attenuated cholera vaccine to model the response to V. cholerae exposure. In this study, we found that vaccination induced plasmablasts which were specific for the two immunodominant antigens of cholera, lipopolysaccharide (LPS) and cholera toxin (CT). These antigens induced a preferential isotype response with LPS driving IgM and IgA plasmablasts while CT drives an IgG and IgA plasmablast. Serum responses had corresponding increases in both antigen binding and functional titers that remained detectable one-year post vaccination. Additionally, LPS specific IgA memory B cells were detectable at 90 days post vaccination. These cells, along with mucosal plasma cells likely play a role in long term cholera immunity. Collectively, these findings demonstrate the use of the live attenuated cholera vaccine as an effective tool to examine the primary and long term immune response to V. cholerae exposure. Additionally, it provides insight into the phenotype and specificity of the cells which likely return to and mediate immunity at the mucosa as determined by our analysis of gut-homing receptor (CCR9) expression levels on antigen specific cells. A thorough understanding of these properties will likely inform future vaccine development to both cholera and other mucosal pathogens.

Lymph node and circulating TFH cells support SIV-specific antibody responses in peripheral blood and genital tract of vaccinated female rhesus macaques

Abstract TFH cells are critical for Ag-specific B cell selection in germinal centers (GCs). Circulating blood CXCR5+ CD4+ T cells (cTFH) were implicated in development of broadly neutralizing antibodies in HIV+ patients. We asked if an SIV vaccine regimen could induce GC TFH and cTFH cells that supported anti-SIV humoral immunity. Female rhesus macaques (RM) were primed twice mucosally with replicating Ad5hr-SIV recombinants (wks0&13) and boosted twice intramuscularly with SIVgp120 proteins (wks26&38). Blood, lymph nodes (LN), bone marrow (BM), vaginal and rectal swabs were collected pre-vaccination, post-2nd prime and post-2nd boost for TFH and B cell assessment. Total TFH, and Env-specific GC TFH cells and Env-specific memory (ESM) B cells were seen at wk38+3d, suggesting vaccine-mediated GC maturation. Env-specific LN TFH cells and GC B cells at wk13+3d correlated respectively with Env-specific rectal IgA and vaginal IgG titers at wk16. GC B cells and ESM B cells at wk38+3d correlated with Env-specific vaginal IgG and rectal IgA titers at wk41 respectively, indicating early GC formation contributed to SIV-specific immunity in the female genital tract (FGT). Env-specific cTFH cells peaked post-boosting and positively correlated with Env-specific IgA and IgG secreting plasmablasts and plasma cells in BM and rectal IgA titers. cTFH cells induced CD38 and CD138 upregulation in autologous B cells and supported IgA secretion in cocultures compared to B cells cultured alone or with non-cTFH cells. Our data indicate the prime/boost regimen induced GC TFH and cTFH cells, enabling robust anti-SIV T-cell-dependent mucosal and systemic B cell responses. Both LN and circulating TFH cells may play pivotal roles in FGT protective humoral immunity.

Antibody and B cell responses to an investigational adjuvanted RSV vaccine for older adults

ABSTRACT Background: Infections with respiratory syncytial virus (RSV) cause significant morbidity and hospitalization in older adults. We studied the humoral, mucosal and B cell responses of an investigational adjuvanted RSV sF vaccine, MEDI7510, in older adults. Methods: In a substudy of a randomized (1:1), double-blind, placebo-controlled study of MEDI7510 in adults ≥60 years of age, we collected blood and nasal secretions at days 0, 8, 29, 91 and 180 post-vaccination to measure F-specific IgG and IgA antibodies by ELISA, and plasmablasts and memory B cells by IgA/IgG dual-color fluorospot. Results: The 27 vaccine- and 18 placebo-recipients had a mean age of 73 years and included 24 women. Among vaccinees, 93% had significant increases in F-specific plasma IgG 85% had increased plasma IgA; 74% had increased nasal IgG and 26% nasal IgA; 93% had IgG and 89% IgA plasmablasts on Day 8 post-immunization; and 82% had IgG and 7.4% IgA memory B cell responses to the vaccine. Vaccinees <70 years of age and women had the highest responses to the vaccine. Conclusions: This adjuvanted vaccine generated robust humoral immune responses in older adults, including RSV F-specific systemic and mucosal antibodies and memory B cells. Nevertheless, age ≥70 years was associated with decreased immunogenicity of the adjuvanted vaccine.

CCL28 Is Involved in Mucosal IgA Responses, Olfaction, and Resistance to Enteric Infections.

CCL28 is a mucosal chemokine that has been involved in various responses, including IgA production. We have analyzed its production in human tissues using a comprehensive microarray database. Its highest expression is in the salivary gland, indicating that it is an important component of saliva. It is also expressed in the trachea, bronchus, and in the mammary gland upon onset of lactation. We have also characterized a Ccl28-/- mouse that exhibits very low IgA levels in milk, and the IgA levels in feces are also reduced. These observations confirm a role for the CCL28/CCR10 chemokine axis in the recruitment of IgA plasmablasts to the lactating mammary gland. CCL28 is also expressed in the vomeronasal organ. We also detected olfactory defects (anosmia) in a Ccl28-/- mouse suggesting that CCL28 is involved in the function/development of olfaction. Importantly, Ccl28-/- mice are highly susceptible to Salmonella enterica serovar Typhimurium in an acute model of infection, indicating that CCL28 plays a major role in innate immunity against Salmonella in the gut. Finally, microbiome studies revealed modest differences in the gut microbiota between Ccl28-/- mice and their cohoused wild-type littermates. The latter observation suggests that under homeostatic conditions, CCL28 plays a limited role in shaping the gut microbiome.

Affinity Maturation Drives Epitope Spreading and Generation of Proinflammatory Anti-Citrullinated Protein Antibodies in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is characterized by the presence of anti-citrullinated protein antibodies (ACPAs); nevertheless, the origin, specificity, and functional properties of ACPAs remain poorly understood. The aim of this study was to characterize the evolution of ACPAs by sequencing the plasmablast antibody repertoire at serial time points in patients with established RA. Blood samples were obtained at up to 4 serial time points from 8 individuals with established RA who were positive for ACPAs by the anti-cyclic citrullinated peptide test. CD19+CD3-IgD-CD14-CD20-CD27+CD38++ plasmablasts were isolated by single-cell sorting and costained with citrullinated peptide tetramers to identify ACPA-expressing plasmablasts. Cell-specific oligonucleotide barcodes were utilized, followed by large-scale sequencing and bioinformatics analysis, to obtain error-corrected, paired heavy- and light-chain antibody gene sequences for each B cell. Bioinformatics analysis revealed 170 persistent plasmablast lineages in the RA blood, of which 19% included multiple isotypes. Among IgG- and IgA-expressing plasmablasts, significantly more IgA-expressing than IgG-expressing persistent lineages were observed (P < 0.01). Shared complementarity-determining region 3 sequence motifs were identified across subjects. A subset of the plasmablast lineages included members derived from later time points with divergent somatic hypermutations that encoded antibodies that bind an expanded set of citrullinated antigens. Furthermore, these recombinant, differentially mutated plasmablast antibodies formed immune complexes that stimulated higher macrophage production of tumor necrosis factor (TNF) compared to antibodies representing earlier time point-derived lineage members that were less mutated. These findings demonstrate that established RA is characterized by a persistent IgA ACPA response that exhibits ongoing affinity maturation. This observation suggests the presence of a persistent mucosal antigen that continually promotes the production of IgA plasmablasts and their affinity maturation and epitope spreading, thus leading to the generation of ACPAs that bind additional citrullinated antigens and more potently stimulate macrophage production of TNF.

Circulating plasmablasts are elevated and produce pathogenic anti-endothelial cell autoantibodies in idiopathic pulmonary arterial hypertension.

Idiopathic pulmonary arterial hypertension (IPAH) is a devastating pulmonary vascular disease in which autoimmune and inflammatory phenomena are implicated. B cells and autoantibodies have been associated with IPAH and identified as potential therapeutic targets. However, the specific populations of B cells involved and their roles in disease pathogenesis are not clearly defined. We aimed to assess the levels of activated B cells (plasmablasts) in IPAH, and to characterize recombinant antibodies derived from these plasmablasts. Blood plasmablasts are elevated in IPAH, remain elevated over time, and produce IgA autoantibodies. Single-cell sequencing of plasmablasts in IPAH revealed repertoires of affinity-matured antibodies with small clonal expansions, consistent with an ongoing autoimmune response. Recombinant antibodies representative of these clonal lineages bound known autoantigen targets and displayed an unexpectedly high degree of polyreactivity. Representative IPAH plasmablast recombinant antibodies stimulated human umbilical vein endothelial cells to produce cytokines and overexpress the adhesion molecule ICAM-1. Together, our results demonstrate an ongoing adaptive autoimmune response involving IgA plasmablasts that produce anti-endothelial cell autoantibodies in IPAH. These antibodies stimulate endothelial cell production of cytokines and adhesion molecules, which may contribute to disease pathogenesis. These findings suggest a role for mucosally-driven autoimmunity and autoimmune injury in the pathogenesis of IPAH.

Mucosal Chemokines.

Several chemokines have important functions in mucosal immunity. While there are many chemokines, 4 of them (CCL25, CCL28, CXCL14, and CXCL17) are especially important in mucosal immunity because they are homeostatically expressed in mucosal tissues. Of these, only CCL25 and CCL28 have been widely recognized as mucosal chemokines. In this study, we review the physiology of these chemokines with specific emphasis on their function in mucosal immunity. CCL25 recruits certain important subsets of T cells that express CCR9 to the small intestine. These CCR9+ T cells also express the integrin α4β7 and have been shown to play important roles in the control of intestinal inflammation. CCL28 recruits CCR10+ IgA plasmablasts to the lactating mammary gland. The role of CXCL14 in mucosal immunity is less well defined, but a Cxcl14-/- mouse exhibits significant metabolic abnormalities. Finally, CXCL17 was the last chemokine to be described and signals through a new chemokine receptor (GPR35/CXCR8), which is expressed in a subset of macrophages that are recruited to mucosal tissues by this chemokine. We conclude that these 4 chemokines play very important roles in mucosal immunity and their continued functional characterization will likely identify novel therapeutic targets.

Alterations on peripheral blood B cell subsets induced by allergic rhinitis.

Here we evaluated whether allergic rhinitis to Dermatophagoides pteronyssinus induces alterations on circulating B cell subsets. Circulating B cell subsets and isotype expression on antigen-experienced B cells from allergic patients under conventional pharmacological treatment (NO-SIT, n = 15) and under subcutaneous immunotherapy (SCIT, n = 33), and non-allergic subjects (NC, n = 25) were analyzed by flow cytometry. In allergic patients, we found a significant decrease in IgM(+) and IgG(+) memory B cells and an increase in IgA(+) memory B cells. Additionally, the numbers of circulating IgA(+) plasmablasts in allergic patients were also increased, while those cells expressing IgM were reduced. Allergic patients have a disturbed B cell subsets distribution which seems to underlie rhinitis pathogenesis and remain unchanged after SCIT.

Impaired migration of IgA-secreting cells to colon adenocarcinomas.

Local inflammation is a strong risk factor for the development of gastrointestinal adenocarcinomas. Mucosal regulatory T cells and IgA-secreting cells both contribute to reduce inflammatory responses, and their recruitment to tissues is dependent on local production of chemokines. More specifically, IgA-secreting cells are recruited to mucosal tissues by CCL28 signalling through CCR10. Here, we examined the recruitment of IgA-secreting plasma cells to tumor-associated mucosa in patients suffering from colon adenocarcinoma. Flow cytometric analyses of single cell suspensions from tumor-associated and unaffected colon mucosa showed a marked decrease in CD19(+)CD38(high)IgA(+) plasmablasts in the tumor-associated mucosa, while the total frequencies of B and T cells were similar. This finding was confirmed in ELISPOT assays, demonstrating a 64% reduction in the frequencies of IgA-secreting cells among cells from the tumor-associated mucosa. The few IgA(+) plasmablasts present in the tumor did not express CCR10, and functional migration assays demonstrated that IgA-secreting cells from tumor-associated mucosa did not migrate in response to CCL28. Taken together, our results show an impaired migration of IgA-secreting cells to colon tumors, presumably caused by a decreased production of CCL28 in the tumor. The lack of local IgA antibodies may lead to impaired barrier function and increased bacterial colonization, driving further inflammatory responses and promoting tumor growth.

Steady state generation of mucosal IgA+ plasmablasts is not abrogated by B cell depletion therapy with rituximab

B cell depletion therapy with rituximab (anti-CD20) has been established for treating RA. While rituximab efficiently removes circulating B cells, its effects on tissue-based B cells, especially on those in...

Expression of Homing Receptors on IgA1 and IgA2 Plasmablasts in Blood Reflects Differential Distribution of IgA1 and IgA2 in Various Body Fluids

Although secretory IgA is the most abundantly produced Ig isotype, the mechanisms underlying the differential distribution of IgA subclasses in various body fluids remain unclear. To explore these mechanisms, we examined the distribution of IgA subclasses, the influence of the nature and sites of encounters with antigens, and the correlation between IgA subclass distribution and homing potentials of circulating IgA plasmablasts. IgA1 predominated in serum, tears, nasal wash fluid, and saliva; the levels of IgA1 and IgA2 were comparable in vaginal wash fluid; and IgA2 predominated in intestinal lavage fluids. Seventy-one percent of circulating IgA plasmablasts secreted IgA1. The intestinal homing receptor (HR), alpha4beta7, was expressed more frequently on IgA2 than on IgA1 plasmablasts, with no differences in the expression of other HRs. IgA subclass distribution among circulating antigen-specific antibody-secreting cells (ASC) was dependent on the nature of the antigen: following vaccination with Salmonella enterica serovar Typhi, unconjugated pneumococcal polysaccharide, or Haemophilus influenzae polysaccharide-diphtheria toxoid conjugate, the proportions of specific IgA1 ASC were 74%, 47%, 56%, and 80%, respectively. HR expression depended on the route of administration: expression of HRs was different after oral than after parenteral vaccination, while no difference was seen between HR expression of antigen-specific IgA1 and IgA2 ASC induced via the same route. The key factors determining IgA subclass distribution in a given secretion are the nature of the antigens encountered at a particular site and the site-specific homing instructions given to lymphocytes at that site. These two factors are reflected as differences in the homing profiles of the total populations of circulating IgA1 and IgA2 plasmablasts.

Sphingosine 1-Phosphate Regulates the Egress of IgA Plasmablasts from Peyer’s Patches for Intestinal IgA Responses

It is well established that Peyer's patches (PPs) are sites for the differentiation of IgA plasma cell precursors, but molecular and cellular mechanisms in their trafficking remain to be elucidated. In this study, we show that alterations in type 1 sphingosine 1-phosphate (S1P) receptor expression during B cell differentiation in the PPs control the emigration of IgA plasma cell precursors. Type 1 S1P receptor expression decreased during the differentiation of IgM(+)B220(+) B cells to IgA(+)B220(+) B cells, but recovered on IgA(+)B220(-) plasmablasts for their emigration from the PPs. Thus, IgA(+)B220(-) plasmablasts migrated in response to S1P in vitro. Additionally, IgA(+) plasmablasts selectively accumulated in lymphatic regions of PPs when S1P-mediated signaling was disrupted by FTY720 treatment. This accumulation of IgA(+) plasmablasts in the PPs led to their reduction in the intestinal lamina propria and simultaneous impairment of Ag-specific intestinal IgA production against orally administered Ag. These findings suggest that S1P regulates the retention and emigration of PP B cells and plays key roles in the induction of intestinal IgA production.

CCR10 expression is a common feature of circulating and mucosal epithelial tissue IgA Ab-secreting cells.

The dissemination of IgA-dependent immunity between mucosal sites has important implications for mucosal immunoprotection and vaccine development. Epithelial cells in diverse gastrointestinal and nonintestinal mucosal tissues express the chemokine MEC/CCL28. Here we demonstrate that CCR10, a receptor for MEC, is selectively expressed by IgA Ab-secreting cells (large s/cIgA(+)CD38(hi)CD19(int/-)CD20(-)), including circulating IgA(+) plasmablasts and almost all IgA(+) plasma cells in the salivary gland, small intestine, large intestine, appendix, and tonsils. Few T cells in any mucosal tissue examined express CCR10. Moreover, tonsil IgA plasmablasts migrate to MEC, consistent with the selectivity of CCR10 expression. In contrast, CCR9, whose ligand TECK/CCL25 is predominantly restricted to the small intestine and thymus, is expressed by a fraction of IgA Ab-secreting cells and almost all T cells in the small intestine, but by only a small percentage of plasma cells and plasmablasts in other sites. These results point to a unifying role for CCR10 and its mucosal epithelial ligand MEC in the migration of circulating IgA plasmablasts and, together with other tissue-specific homing mechanisms, provides a mechanistic basis for the specific dissemination of IgA Ab-secreting cells after local immunization.

CCR10 expression is a common feature of circulating and mucosal epithelial tissue IgA Ab-secreting cells

The dissemination of IgA-dependent immunity between mucosal sites has important implications for mucosal immunoprotection and vaccine development. Epithelial cells in diverse gastrointestinal and nonintestinal mucosal tissues express the chemokine MEC/CCL28. Here we demonstrate that CCR10, a receptor for MEC, is selectively expressed by IgA Ab-secreting cells (large s/cIgA+CD38hiCD19int/–CD20–), including circulating IgA+ plasmablasts and almost all IgA+ plasma cells in the salivary gland, small intestine, large intestine, appendix, and tonsils. Few T cells in any mucosal tissue examined express CCR10. Moreover, tonsil IgA plasmablasts migrate to MEC, consistent with the selectivity of CCR10 expression. In contrast, CCR9, whose ligand TECK/CCL25 is predominantly restricted to the small intestine and thymus, is expressed by a fraction of IgA Ab-secreting cells and almost all T cells in the small intestine, but by only a small percentage of plasma cells and plasmablasts in other sites. These results point to a unifying role for CCR10 and its mucosal epithelial ligand MEC in the migration of circulating IgA plasmablasts and, together with other tissue-specific homing mechanisms, provides a mechanistic basis for the specific dissemination of IgA Ab-secreting cells after local immunization.

Development and maintenance of the gut-associated lymphoid tissue (GALT): the roles of enteric bacteria and viruses.

GALT can be subdivided into several compartments: (a) Peyer's patches (PP); (b) lamina propria (LP); and (c) intraepithelial leukocyte (IEL) spaces. The B-cell follicles of PP are quiescent in neonatal and germ-free (GF) adult mice. Germinal centers (GC), including sIgA+ blasts, appear in the B follicles of formerly GF adult mice about 10-14 days after monoassociation with various gut commensal bacteria. The GC wax and wane over about a 3-week period, although the bacterial colonizers remain in the gut at high density. Neonatal mice, born of conventionally reared (CV), immunocompetent mothers, display GC reactions in PP postweaning, although pups of SCID mothers display precocious GC reactions at about 14 days of life. Normally, gut colonization of neonates with segmented filamentous bacteria (SFB) leads to explosive development of IgA plasmablasts in LP shortly after weaning. Commensal gut bacteria and the immunocompetency of mothers also appears to control the rate of accumulation of primary B cells from “virgin” B cells in neonates. Enteric reovirus infection by the oral route can cause the activation of CD8+ T cells in the interfollicular regions of PP and the appearance of virus-specific precursor cytotoxic T lymphocytes (pCTL) in the IEL spaces. Such oral stimulation can also lead to “activation” of both CTL and natural killer (NK) cells in the IEL spaces. More normally, colonization of the gut with SFB also leads to similar activations of NK cells and “constitutively” cytotoxic T cells.

Determinants of the localization, magnitude, and duration of a specific mucosal IgA plasma cell response in enterically immunized rats.

The origin and fate of specific IgA plasma cells in intestinal lamina propria were studied in rats immunized enterically with cholera toxin (CT). Our major goal was to define how an anti-CT response is focused and sustained at the site of antigen challenge. To distinguish antigen-dependent from antigen-independent mechanisms, CT exposure was restricted to defined portions of intestine and, in some studies, the distribution of antitoxin-containing plasma cells (ACC) was examined in nonimmune adoptive recipients of post-challenge thoracic duct lymphocytes. After enteric priming and challenge, ACC appeared throughout the gut, but were most numerous at the challenged site. About 25% of ACC appearing at the site of jejunal challenge were due to antigen-driven proliferation of memory cells within the lamina propria; the remainder arose elsewhere, apparently in mucosal follicles or mesenteric lymph nodes, and migrated systemically as antitoxin-containing plasmablasts before homing to the lamina propria. The homing of these migrating ACC precursors was not affected by mucosal exposure to CT, nor did they undergo appreciable antigen-driven division after arrival in gut lamina propria. However, homing was specific for the organ from which they arose, i.e., precursors arising from duodenal challenge homed selectively to jejunum, whereas those from colonic challenge homed to the colon. The organ specificity of homing was determined during the challenge response and was independent of the origin of memory cells participating in the response. The survival of migrating ACC precursors did not differ in segments of gut exposed or nonexposed to CT. However, CT exposure at the time of their migration evoked another secondary-type response, due to stimulation of comigrating memory cells, thus sustaining the secondary response at a high level. These results and those in a previous report identify important mechanisms that affect the localization, magnitude, and duration of a specific IgA response, at least in the intestine. These include: 1) organ-specific homing of migrating IgA plasmablasts, 2) antigen-driven generation of IgA plasma cells from memory cells within the lamina propria, 3) enhanced memory at the site of mucosal priming compared to that a distant mucosae, and 4) regeneration of memory cells during the secondary response.